Conceived and designed the experiments: ARB OSS MT LT RO JM JMBJ LØ. Performed the experiments: ARB JM RO JMBJ. Analyzed the data: ARB OSS RO . Contributed reagents/materials/analysis tools: JMBJ HS. Wrote the paper: ARB . Designed software used in analysis: HS.
The authors have declared that no competing interests exist.
Microbial translocation may contribute to the immunopathogenesis in HIV infection. We investigated if microbial translocation and inflammation were associated with innate and adaptive immune responses in adults with HIV.
This was an observational cohort study. Sera from HIV-infected and HIV-uninfected individuals were analyzed for microbial translocation (soluble CD14, lipopolysaccharides [LPS], endotoxin core antibody, and anti-α-galactosyl antibodies) and inflammatory markers (high sensitivity C-reactive protein, IL-6, IL-1 receptor antagonist, soluble tumor necrosis factor receptor II, and IL-10) with enzyme-linked immunosorbent assays. Peripheral blood mononuclear cells (PBMC) from HIV-infected persons and healthy controls (primed with single-stranded HIV-1-derived RNA) were stimulated with LPS, and cytokine production was measured. Finally, HIV-infected patients were immunized with Prevnar 7vPnC±CpG 7909 followed by Pneumo Novum PPV-23. Effects of microbial translocation and inflammation on immunization were analyzed in a predictive regression model. We included 96 HIV-infected individuals, 76 on highly active antiretroviral therapy (HAART), 20 HAART-naive, and 50 healthy controls. Microbial translocation and inflammatory markers were higher among HIV-infected persons than controls. Cytokine levels following LPS stimulation were increased in PBMCs from HAART-naive compared to HAART-treated HIV-infected persons. Further, RNA-priming of PBMCs from controls acted synergistically with LPS to augment cytokine responses. Finally, high serum LPS levels predicted poor vaccine responses among HAART-naive, but not among HAART-treated HIV-infected individuals.
LPS acts synergistically with HIV RNA to stimulate innate immune responses
Untreated HIV infection is characterized by progressive immune dysfunction. In the first weeks of infection, immense CD4+ T cell depletion and active viral replication occur, particularly in the intestinal mucosa
Lipopolysaccharides (LPS) and LPS-related markers such as soluble CD14 (sCD14) have been used to quantify microbial translocation in peripheral blood
This was an observational cohort study. Patients were recruited from a vaccination cohort consisting of otherwise healthy HIV-infected adults immunized with pneumococcal vaccines with or without CPG 7909
Microbial translocation was investigated by quantification of sCD14, LPS, endotoxin core antibody IgG (endoCAb) (Hycult biotech), and anti-α-galactosyl antibodies (anti-Gal IgM and IgG) in serum samples. Inflammation was quantified by measuring serum levels of soluble tumor necrosis factor receptor II (sTNF-rII), interleukin-1 receptor antagonist (IL-1Ra), high sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and interleukin-10 (IL-10) by enzyme-linked immunosorbent assay (ELISA) (RnD Systems and Invitrogen, DK). LPS levels were evaluated with a chromogenic limulus amebocyte lysate assay (LAL assay, QCL-1000®, Lonza, DK). The anti-Gal antibody assay was an in-house time-resolved immunofluorometric assay (TRIFMA). Briefly, plates were coated with 100 µL/well α-galactosyl-human serum albumin (α-Gal-HSA) glycoconjugates (Dextra, UK) or HSA (0.25 µg/mL), incubated in a humidifier over night at 4°C, and then blocked one hour with 1 mg HSA/mL tris-buffered saline (TBS). Next, 100 µL/well diluted serum samples, controls, blanks, and standard curve samples were added in duplicate. Following an over night incubation, biotinylated anti-human-IgG or –anti-human-IgM antibodies were added (100 µL/well, 0.2 µg/mL). After one hour, streptavidin-Eu was added to the plate. After an additional hour, Enhancement solution (PerkinElmer) was added. Counts per second were measured for each well on a Victor 3 Multilabel Reader (PerkinElmer). The relevant anti-HSA signal was subtracted from anti-α-Gal-HSA signals. Corrected signals were converted to measures of antibody concentration by relating them to a standard curve (the antibody concentration of standard curve samples with dilutions similar to samples were assigned a value of 100 AU). All other assays were performed according to the manufacturer's protocol. Up to three freeze-thaw cycles were performed on serum samples. Median intra-assay and inter-assay coefficients of variation for all markers were below 15%. All serum measurements were performed on pre-vaccinated samples.
The impact of HIV RNA and LPS on pro-inflammatory cytokine secretion was investigated in PBMCs from 20 HAART-treated and 20 HAART-naive individuals with HIV (with matched CD4+ cell count, age, and sex) with PBMCs exposed to one freeze-thaw cycle. For 48 hours, the PBMCs were either stimulated with 100 ng/mL LPS (Invivogen, France) or left untreated. A 25-plex cytokine luminex assay (Invitrogen, DK) was used to measure a large range of cytokines in harvested cell culture supernatants for instance TNF-α, IFN-α, and IFN-γ.
The co-stimulatory effect of viral RNA on LPS-induced cytokine production was further investigated in PBMCs from HIV-uninfected controls, which were exposed to one freeze-thaw cycle. PBMCs were either primed by transfection with increasing concentrations of HIV-1-derived single-stranded RNA (ssRNA40: 0, 0.01, 0.1, or 1.0 µg/mL) or unrelated control RNA (ssRNA41: 0.1 µg/mL) (both Invivogen, France). After incubation for 24 hours, PBMCs were stimulated with LPS at increasing concentrations (0, 1, 10, or 100 ng/mL). Supernatants were harvested and stored at −80°C. The pro-inflammatory marker TNF-α was measured in cell culture supernatants using a Cytoset ELISA (Invitrogen, DK).
All HIV-infected subjects were immunized, and all together three doses of vaccine were given. First with double the standard dose of 7-valent pneumococcal conjugated vaccine (7vPnC, Prevnar®, Wyeth) ±adjuvant (1 mg CpG 7909, Pfizer) at 0 and 3 months and with one single dose of 23-valent polysaccharide vaccine (PPV-23) (Pneumo Novum®, Sanofi-Pasteur MSD) ±1 mg CpG 7909 at 9 months
Immune responses to pneumococcal vaccination were quantified by specific anticapsular IgG levels for all seven 7vPnC vaccine serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F)
HIV-infected individuals were categorized as “HAART-naive” if they had never been exposed to antiretroviral drugs or “HAART-treated” if, at inclusion, they received either a 3-drug regimen including a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, and/or abacavir or a 2-drug combination of a non-nucleoside reverse transcriptase inhibitor and a boosted protease inhibitor. All HAART-treated HIV-infected individuals had been treated for at least six months and had undetectable viral load at enrollment. HIV-uninfected persons were referred to as “controls”.
Medians and interquartile ranges (IQR) were calculated for all continuous variables. Sensitivity analyses were conducted to evaluate if participants, who on a single occasion demonstrated markers with extreme values, on their own caused the observed differences. Kruskal-Wallis rank test was used for comparison of more than two variables. Student's t-test was applied to test differences in the markers between groups. Logarithmic transformations (log10) were made when continuous outcomes did not follow a normal distribution. When logarithmic transformed values did not follow a normal distribution, Mann-Whitney rank sum was used. Spearman's correlation was used to evaluate correlations between markers.
LPS-responsiveness in PBMCs from HIV-infected individuals was evaluated by a stimulation index as the ratio between secreted cytokines from LPS-stimulated and untreated PBMCs. The measure of adaptive immune response was an aggregated outcome based on measurements of vaccine-specific antibodies (at 0, 3, 4, 9, and 10 months). To account for missing data, we employed a multiple imputation strategy
The study included 96 HIV-infected individuals (20 HAART-naive and 76 HAART-treated) and 50 controls. Pre-vaccination characteristics and laboratory results for all subjects are shown in
HAART-treated (n = 76) | HAART-naive (n = 20) | Controls (n = 50) |
|
|
Age, years |
48.9 (42.8–59.9) | 48.2 (40.6–55.3) | 41.3 (31.3–51.3) | <0.0008 |
Sex, male, n (%) | 65 (85.5) | 16 (80) | 30 (60) | = 0.004 |
BMI index |
23.5 (21.7–25.2) | 24.6 (22.6–26.5) | - | = 0.19 |
CD4+ cell count, cells/µL | 641 (489–837) | 497 (373–812) | - | = 0.25 |
CD4 nadir, cells/µL | 201 (71–243) | 401 (313–659) | - | <0.0001 |
Log10 HIV RNA | 1.60 (1.60–1.60) | 4.33 (3.72–4.70) | - | <0.0001 |
Current smokers, n (%) | 26 (34.2) | 9 (45) | - | <0.001 |
sCD14, µg/mL | 6.57 (4.85–9.39) | 7.24 (5.07–11.4) | 3.12 (2.76–3.66) | <0.0001 |
LPS, EU/mL | 1.62 (1.28–2.07) | 1.71 (1.49–1.98) | 1.43 (1.14–1.88) | = 0.16 |
EndoCAb, GMU/mL | 27.3 (15.4–51.7) | 24.5 (17.1–36.5) | 34.6 (25.4–56.9) | = 0.04 |
Anti-Gal |
178 (89.4–386) | 140 (78.4–540) | 152 (78.3–564) | = 0.83 |
Anti-Gal IgG, AU | 9.28 (3.81–23.1) | 9.30 (6.09–24.2) | 13.38 (6.91–27.2) | = 0.30 |
sTNF-rII, ng/mL | 5.0 (3.6–6.2) | 5.8 (4.6–8.4) | 4.3 (3.7–4.8) | <0.0002 |
IL-1Ra, pg/mL | 66 (39–104) | 88 (70–117) | 67 (47–93) | = 0.044 |
hs-CRP, µg/mL | 5.47 (2.18–13.0) | 5.42 (1.79–8.15) | 2.49 (1.00–7.52) | = 0.01 |
IL-6, pg/mL | 0.32 (0.12–0.60) | 0.44 (0.28–0.78) | 0.13 (0.09–0.37) | = 0.006 |
IL-10, pg/mL | 1.49 (0.77–2.27) | 2.88 (1.79–3.87) | 2.07 (1.13–3.86) | <0.001 |
Global p-value, Kruskal-Wallis rank test.
Unless otherwise indicated the median (IQR, interquartile range) is given.
BMI, body mass index.
Anti-Gal, anti-α-galactosyl.
AU, arbitrary units.
Soluble CD14 levels were significantly higher in HIV-infected subjects (6.58 µg/mL, IQR: 4.88–9.47) than in controls (3.12 µg/mL, IQR: 2.76–3.66, p<0.001), and there was a trend towards higher LPS in HIV-infected subjects (1.67 EU/mL, IQR: 1.30–2.06) compared to controls (1.43 EU/mL, IQR: 1.14–1.88, p = 0.06) (
(
The levels of the pro-inflammatory markers, sTNF-rII and IL-6, were significantly higher in HAART-treated and HAART-naive individuals compared with controls (
Following LPS stimulation of PBMCs, greater stimulation indexes were observed in HAART-naive subjects compared with HAART-treated subjects for the main pro-inflammatory cytokines, TNF-α (p = 0.03), IFN-α (p = 0.002), and IFN-γ (p = 0.003) (
(
In PBMCs from HIV-uninfected controls, we found higher TNF-α responses in cells primed with HIV-1-derived single-stranded RNA (ssRNA40) (n = 4) compared with un-primed and unrelated ssRNA41-primed PBMCs after LPS stimulation (
In HAART-naive subjects, LPS levels predicted the total specific IgG antibody response to pneumococcal vaccine ( The predictive estimate of soluble markers for microbial translocation and pro-inflammation on adaptive immune response after pneumococcal vaccine in HAART-treated and HAART-naive HIV-infected individuals demonstrated by estimate and 95% confidence interval. LPS was found as an independent predictor after adjustment and mulitiple comparison. Adjustment for current smoking status, pre-vaccinated CD4+ cell count, age, HIV RNA (log10), and ±TLR9-agonist in the pneumococcal vaccine.HAART-treated (n = 76) HAART-naive (n = 20) Estimate, unadjusted Estimate, adjusted Estimate, unadjusted Estimate, adjusted sCD14 −0.62 (−1.44–0.20) −0.41 (−1.23–0.41) −0.10 (−1.49–1.29) −0.26 (−2.11–1.58) LPS 0.51 (−0.37–1.39) 0.75 (−0.16–1.65) −0.99 (−2.41–0.42) −2.62 (−4.06–−1.17) EndoCAb 0.002 (−0.003–0.007) 0.001 (−0.004–0.006) −0.003 (−0.01–0.006) −0.003 (−0.01–0.007) Anti-Gal IgM 0.16 (−0.20–0.52) 0.08 (−0.28–0.43) 0.20 (−0.19–0.59) 0.46 (−0.13–1.04) Anti-Gal IgG −0.04 (−0.29–0.21) −0.03 (−0.28–0.21) −0.09 (−0.48–0.29) −0.07 (−0.67–0.52) sTNF-rII 0.05 (−0.79–0.89) 0.35 (−0.48–1.19) −0.21 (−2.01–1.58) 0.05 (−2.33–2.42) IL-1Ra 0.27 (−0.22–0.77) 0.20 (−0.29–0.69) 0.11 (−1.42–1.64) 1.04 (−1.04–3.12) IL-6 −0.35 (−0.70–−0.01) −0.28 (−0.65–0.08) −0.33 (−0.87–0.22) −0.18 (−0.88–0.51) hs-CRP −0.15 (−0.45–0.14) −0.05 (−0.35–0.24) −0.23 (−0.84–0.38) −0.05 (−0.93–0.83) IL-10 0.19 (−0.26–0.63) 0.20 (−0.25–0.65) 0.14 (−0.48–0.75) 0.32 (−0.54–1.17)
In this study, we evaluated the independent impact of microbial translocation and pro-inflammation on innate and adaptive immune responses. Interestingly, we found an inverse relation between baseline serum LPS and subsequent adaptive immune response in HAART-naive individuals. This association was not observed among HAART-treated subjects. We also found the release of pro-inflammatory cytokines after LPS stimulation was increased in PBMCs from viremic HAART-naive subjects compared to HAART-treated subjects, as well as in PBMCs from healthy controls pre-treated with HIV-1-derived RNA, and similar findings have been demonstrated in previous studies
Anti-Gal immunoglobulins are potential novel markers for microbial translocation and HIV infection. Specific anti-Gal antibodies make up approximately 1% of circulating IgG antibodies
We did not retrieve any association between endoCAb and LPS or endoCAb and sCD14, while another study found an inverse correlation to LPS
This study had some limitations. A cross sectional study design has obvious limitations due to lack of follow up. Participants in our HAART-naive group consisted of a relatively small number compared to other studies on microbial translocation
No significant association was found between LPS and sCD14 in our study, which is in accordance with findings of some studies
LPS is the only primary marker of microbial translocation measured in our study, since sCD14 and endoCAb are host markers. Another primary marker, which might be relevant to measure, could be bacterial ribosomal 16S RNA (16S rDNA), which is found higher in HIV-infected individuals compared to uninfected, and 16S rDNA correlates with LPS
In this study we used samples from a vaccination cohort. Protocol samples were collected at three immunizations and at follow-up
The innate toll-like receptors (TLRs) recognize pathogens and, upon activation, induce and direct immune responses. HIV single-stranded RNA, a TLR7- and TLR8-ligand
Pneumonia is more common in HIV-infected individuals with a 6-fold higher incidence than in HIV-uninfected individuals
It is important to further investigate pneumococcal vaccination effectiveness in HIV-infected subjects in relation to microbial translocation and innate immune function. If our findings translate in to clinical effectiveness, then increased LPS levels at the time of immunization would independently predict vaccine failure in HAART naive individuals.
There is a great deal of uncertainty regarding the relationship between HIV disease progression and microbial translocation and subsequent inflammatory immune responses
In conclusion, we found LPS to be an independent predictor of adaptive immune response in untreated HIV-infected individuals. Our results suggest HIV RNA and LPS act in synergy and that their concerted action brings about increased cytokine responsiveness through innate immune recognition pathways. By reducing viremia, HAART treatment increases the tolerance to LPS, which in turn reduces further immune activation. Hence, earlier initiation of HAART may offset the immune disruption caused by microbial translocation and should be investigated in future studies.
PBMC responsiveness to HIV RNA and LPS. (
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Flowchart from main trial.
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Pneumococcal CPG7909 protocol from main trial.
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The authors thank the participants in the pneumococcal vaccine study. They also thank Kirsten Stadel Petersen and Søren Riss Paludan (Department of Medical Microbiology and Immunology, Aarhus University, Denmark), for luminex measurements, and the blood bank at Aarhus University Hospital, Skejby, Denmark, for collecting blood samples from HIV-uninfected controls.